JPS61210040A - Medical collagen membrane - Google Patents

Abstract

Collagen membranes with desired properties are prepared by using a variety of gel-forming techniques in combination with methods for converting the gels to solid forms. One technique involves compressing a gel matrix under constant pressure to form a fibre network which is dried; another involves disrupting and centrifuging a matrix, harmonizing the precipitate auto or paste and casting and drying the paste. The properties of these membranes or other solid forms may be further altered by cross-linking the collagen preparation either after formation of the membrane or gel, or most preferably by mixing cross-linked collagen with solubilized collagen in the original mixture used to create the gel.

Description

DETAILED DESCRIPTION OF THE INVENTION TECHNICAL FIELD This invention relates to the field of materials useful for tissue repair and related to wound treatment. More particularly, the present invention relates to a collagen membrane material produced by a novel method, the layer being biocompatible, non-inflammatory, and useful for tissue repair as an artificial implant. .

BACKGROUND OF THE INVENTION Numerous attempts have been made to obtain artificial membranes that can be used as replacements for skin, blood vessels, ligaments or other connective tissue. Collagen is usually the main component of connective tissue, so many of these membranes use collagen. Collagen only (e.g., U.S. Pat. No. 44129
No. 47; Japanese Patent No. 741 (No. 139174; and US Pat. No. 4,242,291) or collagen in combination with other materials (e.g., US Pat.
There is a large body of literature on methods for producing such membranes. Other membranes include combinations of substances such as glycoproteins with fibrinogen and thrombin (European Patent Application No. 92200) and combinations of keratin-derived polymers and gelosaminoglycan polymers (European Patent Application No. 891).
No. 52) is used.

The sexual properties and quality are determined by the nature of the material, such as the nature of the collagen used to form the membrane and the method used to form it. Conventional membranes are used in corneal substitutes, artificial skin and wound treatment. It has met with some success in its intended uses, including: However, many cause inflammation and have less than optimal properties in terms of flexibility, biological stability and strength.

The present invention imparts desired properties in the resulting membrane by using non-immunogenic collagen formed into a membranous material by a series of methods that impart flexibility in the physical properties of the product. , provides a way to adapt these properties to the intended use. The membranous material can be used as a two-dimensional membrane, including membranes that can be formed into tubular containers, three-dimensional implants, or one-dimensional fibers.

DISCLOSURE OF THE INVENTION The present invention provides that the physical properties of collagen membranes make them suitable for use in a variety of medical applications, including vascular repair, ulcer repair, luminal surface reconstruction, skin repair, and artificial skin. It provides a collagen membrane. The layer can also be used as a substrate for the production of desired cell cultures in vitro. The properties of the layer are determined by appropriate selection from a range of manufacturing methods to obtain properties suitable for the chosen application. Similar flexibility can be obtained in the properties of one-dimensional and three-dimensional structures produced by modifications or additions to the membrane manufacturing method.

The resulting fibers are useful as ligament or ligament substitutes, and can also be used as three-dimensional blocks or solids to provide sutures, implants for use in tissue repair or wound filling.

That is, in one aspect, the present invention optionally provides a general method of mixing a solution of atelopeptide collagen and a suspension of crosslinked collagen to obtain a gel from the solution, and converting the gel into a membrane form. Collagen membranous material produced by the method) In other embodiments, the invention relates to fibers or solids made from the gel.

In yet another aspect, the present invention relates to the general method itself and specific methods used within the general method to obtain two-dimensional membranes, fibers, and solids of desired properties.

Gels can be obtained from solubilized collagen or mixtures by three different methods. In one method, a solution of collagen is treated with a precipitation buffer that insolubilizes the collagen by increasing the pH. In this method, a gel is formed by cooling, mixing, and then warming to about room temperature. In a second method, the mixture of collagen and buffer is centrifuged, the supernatant from the centrifugation is collected, and the mixture of collagen and buffer is warmed to approximately room temperature instead of being warmed up using gravity pressure. In a third method, a solution of collagen is treated with an insolubilizing solution at room temperature. The insolubilization solution is prepared such that the resulting mixture is at physiological pH and ionic strength. This mixture is then incubated at approximately 37°C to form a gel. The third method can be modified to degas the mixture immediately after mixing and to form the degassed mixture before incubation. The method in each of the three cases described above can also be applied to the formation of gels containing suspensions of crosslinked forms in addition to dissolved collagen. The presence of this additional crosslinking substrate further alters the properties of the membrane material ultimately obtained from the method of the invention.

The above gel formation method for containing crosslinked collagen in the starting material includes formation of a gel from only solubilized collagen, and then partial crosslinking or crosslinking of the layer after film formation from the gel makes it more brittle; Considered critical in the sense that it is considered to result in an unsatisfactory product.

Alternatively, if the method for forming a gel from solubilized collagen is modified by adding a crosslinking substance described below, the third method described above, i.e., a solution of collagen (containing the desired crosslinking substance in the mixture). At room temperature, the mixture is at physiological p
A method of treatment with an insolubilizing solution prepared to have a high ionic strength of H and ionic strength is preferred. The mixture is incubated at about 37°C to form a gel. In particularly preferred embodiments, the mixture is degassed before adding the insolubilizing solution.

The conversion of the gel into a membrane can also be carried out by two other basic methods. In one method, the gel is compressed under constant pressure to form a mat and then dried. When using this method, in addition to obtaining two-dimensional membranes, solid implants can be prepared directly by compressing the shaped gel obtained from a modification of the gel formation method using degassing. A fibrous product is obtained by applying pressure around a cylinder formed from gel. In the second method, the gel is crushed, the crushed gel is centrifuged to obtain a precipitate, and the precipitate is cast and dried. Depending on the size and shape of the mold,
Either membranes or solids can be obtained.

Description of drawings Figure 1 and! @2 Figure shows the prepared membranes G-2 and G-
This is an electron micrograph of 3, magnified 30,000 times.

Embodiment A of the Invention, Preparation of CIS The method of the invention starts with either collagen in solution or in a mixture with cross-linked fibrillar collagen.

Collagen can be solubilized and purified from mammalian connective tissue;
Prepared from bovine skin, pig skin and mammalian bone as well as many other sources. The purification method is publicly disclosed, for example, US Pat. No. 3,949,073 and US Pat. No. 4,066,083.
No. 8 and British Patent Publication No. 1565340. Collagen can be easily solubilized to concentrations useful in the present invention by acid dissolving the fibers in a manner similar to the stated method, and dissolves at a pH of 1 to 4. In fact, collagen in solution (C15) is manufactured by Collagen Co., Palo Aldo, California.
poration, Pa1n Alto, Nioli
Fornia is conveniently available commercially under the trademark Zygen.

Natural collagen exists in the form of fibers consisting of a triple helical structure of peptide chains. The helical structure results from the repeating of a triple sequence composed of two amino acids, usually glycine linked to proline and hydroxyproline in the amino acid sequence. These regions of triple repeating units are assembled into a triple helical structure. Furthermore, all collagen chains do not have triple glycine sequences at each end and therefore contain regions that are not helical. These regions are thought to be responsible for the immunogenicity associated with most collagen preparations and are termed telopeptides. The immunogenicity of collagen preparations can be alleviated primarily by the removal of these telopeptides, resulting in "atelopeptide collagen." Removal of telopeptides is accomplished by digestion with proteolytic enzymes such as trypsin or pepsin. The non-helical telopeptide region also needs to form crosslinks, which are responsible for the stability of the fibrous structures in natural materials. If it is desired to impart this property, the atelopeptide collagen must be artificially crosslinked.

The collagen solution, which is the starting material for the method of the present invention, is
Atelopeptide collagen, preferably a dilute commercial product such as Chigen CIS.

Collagen concentrations ranging from 1 to 1 ONfl/me are suitable for use in the present invention. Of course, while this range is suggestive of suitable concentrations, it is not meant to be an absolute limitation, and the upper and lower limits are also arbitrary within the scope of the invention.

B. Preparation of Cross-Linked Collagen As used herein, "cross-linked collagen" is defined as a compound whose viscosity, as measured by chemical or radiation treatment or at 22° C. and a shear rate of 5000 sec. Reconstituted atelopeptide collagen is shown that has been artificially cross-linked and purified by other suitable means to obtain sufficient cross-linking of 3000 centipoise.It should be noted that the above limitations are arbitrary and illustrative of useful ranges.

Prepare the crosslinked form. For this purpose, solubilized collagen is first neutralized and precipitated at room temperature or
Zyderm) using preparations of precipitated or reconstituted collagen, such as collagen implants, and subsequent reactivity to chemical crosslinking agents such as formaldehyde, glutaraldehyde, glyoxal, etc. or reactivity to ionizing radiation such as gamma rays. Crosslinking using standard methods including. Heating and UV irradiation can also be used, but are less effective. The cross-linked material is then collected by centrifugation, washed with a suitable aqueous solution, eg, physiological saline, and concentrated to a workable suspension concentration of 1-10 mg/gl.

More specifically, a cross-linking agent is a polyfunctional compound that is used at a concentration to produce viscous collagen that is covalently cross-linked before being quenched with an agent that forms a non-fouling, water-soluble adduct with the cross-linking agent. Polymers, and more commonly polyesters, are difunctional compounds. The concentration of collagen in the suspension during the reaction, the concentration of the crosslinking agent and the duration of the crosslinking reaction are important and depend on the nature of the crosslinking agent. Collagen concentration typically ranges from 0.1 to 10/xi, more typically from 1 to
The range is 5~/ml (7). Aldehydes are preferred as crosslinking agents; suitable aldehydes include formaldehyde, glutaraldehyde, acid aldehydes, glyoxalbilvic aldehyde and aldehyde starch, preferably glutaraldehyde. Amines are preferred as quenching agents, and glycine is particularly preferred. If glutaraldehyde is selected as the crosslinking agent, its concentration is typically about 0% by weight/volume.
．． 0.001% to 0.05%, and the crosslinking reaction proceeds over about 0.5 hours to 1 week. The reaction is carried out with the crosslinking agent in at least stoichiometric proportions, but preferably in excess, before the quenching agent is removed. 1
One typical cross-linking experimental method involves a collagen concentration of 3
q/ml and glutaraldehyde 0.01 grave regrets,
Examples include 16 hours at 22°C. The crosslinked product is washed to remove unreacted crosslinker, polymer formed by the crosslinker, and unreacted quenching agent if quenched. A buffer that can be used is a sodium phosphate/sodium chloride buffer with a pH of about 7.

The wash product is typically washed by about 20-50%/III/%, preferably about 25%-
401111! /ml can be concentrated to an appropriate protein concentration range. The cleaning product contains approximately 20 ppp of aldehyde.
Vibrating plate viscometer (Nametre Co., Ltd.) that measures dynamic viscosity, not steady flow.
The viscosity should be in the range of about 700 to 3000 CP at 22° C., as measured on a PBD, model 7.006PBD).

C. Gel Formation The method of the present invention for forming collagen membranes or related membranous materials essentially involves the formation of a gel from collagen in solution and the conversion of the gel into a membrane or other desired form. It consists of two steps.

Each of these steps has a range of temperatures, gravity and properties. The temperature at which gel formation is carried out can be from about 100° C. to about 37° C., the ionic strength can vary from about 0.05 to about physiological ionic strength, and the gravity field conditions can range from about IXy to about 13° C.
It can be changed between 000xf. The typical method shown below represents a representative example of these changes, and intermediate cases may also be used.
It is also useful and depends on the desired membrane properties.

It is believed that degassing and shaping prior to gel formation results in a tougher product that can be further processed and formed into fibers, films, or solids.

Three general methods can be used as outlined above. First, the CIS is cooled to an ionic strength well below physiological ionic strength, preferably about 0.05
and pre-chilled buffer solution at an ionic strength of 0.000000000000 and incubating the mixture at low temperature. Second, the CIS is mixed with a buffer at physiological ionic strength and temperature conditions and incubated at physiological temperature. According to the third method,
The CIS is treated with buffer as in the first method, except that it is subjected to gravitational pressure before incubation at room temperature.

The gel may optionally contain any amount of the crosslinked collagen material prepared from solubilized collagen. The relative amounts of crosslinked and non-crosslinked collagen material and the conditions of the gel and membrane formation steps are believed to determine the properties of the resulting membrane. Any similar equipment may be used to form the gel and convert it into a membrane, whether or not a crosslinking material is included in the original composition.

In the formation of gels, in the case of intermediate materials for the final membranous product containing cross-linked fibrous collagen, the gelling process includes collagen in solution as well as suspensions containing cross-linked forms at the suspension concentrations mentioned above. It is carried out as described above, except that a portion of the liquid is mixed with solubilized collagen. The ratio of the two components, based on the relative weight of the collagen they each contain, varies considerably depending on the desired end product properties; Higher ratios are used, and higher concentrations of crosslinked forms are used to obtain tougher final product films. For example, if the final membrane product is used to hold sutures as an artificial spear or to form a tube and perform such suture connections, approximately 40-80% cross-linking Collagen ratio 1 is preferably about 50%.

If the original mixture for gel formation contains both a suspension of crosslinked collagen and collagen in solution, the composition of any film formed is the weight of crosslinked collagen relative to the total collagen content! If the membrane is described as containing 10% cross-linked collagen,
The original component is supplied with a cross-linked collagen suspension containing 10% of the total weight of collagen and 90% as soluble solution collagen. Compositions of the invention can contain up to 90% crosslinked collagen.

D. Conversion into Membranes Conversion of gels into membranes is accomplished by compressing the gel to squeeze out the liquid to form a more intimate "mat" and then drying it at about atmospheric pressure, e.g. in air. Alternatively, any fundamentally different method can be obtained by crushing the gel matrix, centrifuging the crushed material to recover precipitated collagen, homogenizing the precipitate into a paste, and placing the paste in a mold. The properties of the resulting membranes that can be achieved with the two methods mainly depend on which of these two conversion methods is used, with the product of the compression method being soft, transparent and clear, and relatively Forms a film-like material with high tensile strength. The product of gel fragmentation and subsequent precipitation of the fragments is relatively friable, translucent, has a rough surface, and is relatively thick.

However, both films have a diameter of about 70 to 3 Q Q nm.
, can be characterized as a random fibrous network of fibers approximately 0.5-5μ in length.

The inclusion of cross-linked collagen does not change these properties other than making the product tougher. The crosslinking material is the fibrous network □
incorporated into the organization.

In the compression method, for example, it is pressed in a conventional piston-type device, such as the device currently used to obtain tofu.

Compression is performed at about room temperature by using the collagen gel in the medium in which it is prepared.

Compression is performed using 1.1 to 3 atmospheres and continues until the volume is less than about 5% of the original gel. The resulting flat collagen fiber mat is then dried at low temperatures (below about 37° C.) in air or other suitable atmosphere to obtain the desired T membrane. Additionally, it is desirable to flush residual T salts from the membrane. Cleaning can be accomplished by washing with water, again
Dry at low temperature under atmospheric pressure.

Methods using gel fragmentation are typically carried out by mechanically fragmenting the matrix, e.g. using a spatula, followed by approximately 8000-16000 x g
, preferably at about 13000XF for about 20-30 minutes to obtain a precipitate. The centrifugal branch force can of course be changed and
A clear limited area cannot be set. The precipitate is then thoroughly homogenized at room temperature to form a paste-like substance, and the paste is placed in a mold and solidified at low temperature (below about 37° C.) and atmospheric pressure. The dried material is then desalted by washing with water and dried again, if desired.

Although crosslinking of the collagen in the resulting membranous material is optional, crosslinking can be carried out by treating the membranous material with glutaraldehyde to obtain a desired crosslinked product. However, as mentioned above, this method results in the production of a more brittle product. This crosslinking method is known. In summary, in a typical method, the substance has a concentration of 0.05
1-16 in a solution containing ~1% glutaraldehyde
time, and then the glycine concentration was about 0.1-0.4M.
Quench by adding glycine solution until

The crosslinking solution is then removed by washing.

E. Applications The resulting materials can be used in soft tissue repair configurations, usually using artificial membranes, such as, for example, burn skin replacement, key reconstruction or wound repair. They can also be molded into various shapes and used with hard tissue replacement. The molded or compressed membrane can be reshaped into a three-dimensional object for implantation at a bone replacement site by cylindrical or stacked into a mold and cut. The layer can also be used in a two-dimensional configuration to continuously implant the membrane into a defect site, such as a cranial or periodontal sinus. Generally, onlay-type repairs can be performed by stacking these membranes over the sinus.

Three-dimensional implants can also be obtained directly from the gel by compacting it in a suitable mold.

In this method of construction, the mixture containing CIS and precipitation buffer is preferably degassed and placed in a mold before compression. (Degassing can also be used in related methods to make membranes and fibers.) Degassing and drying and cleaning the dense collagen fiber network formed by compression of the collagen gel into the mold Desalted by
Before re-drying, it is put into the mold again and aged at high temperature,
Promote remaining crosslinking. Furthermore, the fibers preferably
Can be formed directly from the gel before compaction or crushing.

The gel is wrapped in a porous adsorbent and squeezed or rolled into desired II diameter fibers. Shredded gels can also be used, but in this case the lam must be formed by casting and stretching, making the process cumbersome and difficult to achieve the desired product.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these. The first three examples show different gel formation methods combined with a compaction method for film formation - Examples 4-6 also show a similar gel formation method followed by film formation using crushed material. There is.

All of the above is based solely on CIS. Examples 7 and 8 demonstrate the formation of crosslinks in membranes obtained either by compression or by fragmentation and precipitation recovery. Example 9 shows the use of a degassed and shaped mixture to form a gel, which is used directly to form a three-dimensional implant. Example 10
shows the formation of a gel containing partially cross-linked collagen, and Example 11 shows the conversion of this gel into a membrane. Example 12 shows another example of a membranous material containing cross-linked collagen.

Example 1 Chigen(2)q//tttl bovine atelopeptide collagen, in hydrochloric acid, pH 1-4) CIS9011 at 4°C
of pre-chilled 0,2 M Na2HP 0
Mix with 10 Ill of buffer containing 410.09M NaOH. The solution is mixed at 4°C and incubated at room temperature for about 16-20 hours, conveniently overnight. The resulting collagen gel is then placed in a press and compressed into a flat collagen fiber network using low pressure of about 1.5 atmospheres. The resulting network is air-dried at room temperature, washed with water, and air-dried again. The obtained collagen membrane is designated as G-1.

Example 2 Digen CICl39O at room temperature was dissolved in 0.2 MNa2 at room temperature.
HP O4/ 1.3 M NaC (1/ o, 09
Mix with 10 liters of M NaCIF (containing buffer) and incubate the mixture overnight at 37°C. The resulting matrix is converted into a membrane as described in Example 1. The resulting membrane c-2 is It is a smooth, flexible and transparent substance. An electron micrograph of its fiber structure is shown in Figure 1.

Example 3 Chian Cl59Q was cooled to 4°C and 0.2MNaz
HP O47a, o 9 MNaOHff refrigerated (4℃
) Mix quickly with 10 +++ l of buffer and immediately transfer to a centrifuge bottle. The mixture is centrifuged at 8θ00×y for 2 hours at approximately 20° C. and the supernatant is collected from the bottle. Incubate the supernatant overnight at 20°C to obtain a gel. The gel was converted into a membrane exactly as described in Example 1 and designated as G-3. An electron micrograph of the fiber structure is shown in FIG.

Example 4 Tigen Cl590at/1 and insolubilization buffer 10+1 are mixed at 4°C and incubated to form a gel exactly as described in Example 1. Crush the gel matrix with a spatula, transfer to a centrifuge bottle, and incubate at 130'00.
Centrifuge for 30 minutes at XI. The resulting precipitate is collected and homogenized into a paste. Take the paste into a mold,
Air-dry at 37°C, then wash with water, air-dry again at 37°C,
A membrane p-1 is obtained.

Example 5 Tigen CIS was treated with a buffer to form a gel exactly as described in Example 2, and the gel was then treated with a buffer as described in Example 4.
Convert it into a membrane using exactly the same method as shown in .

The obtained membrane is designated as P-2.
A gel is formed using S and the resulting gel is converted into a membrane as shown in Example 4.

The obtained membrane is designated as P-3. Example 7
Na2HP O4/ 1.3 M NaC1/ 0.0
Mix with 10 ml of 9M NaOH-containing buffer and incubate the mixture overnight at 37°C. The resulting gel is compressed, dried, washed and desalted as described in Example 4. The washed membrane was then dissolved in water at 20°C.
% glutaraldehyde, the crosslinked membrane is washed and dried at low temperature to obtain membrane XG-2.

Example 8 A gel is formed from Tigen Cl590M1 in the same manner as in Example 7, and the resulting gel is crushed with a subachera, transferred to a recirculation bottle, and centrifuged at 13,000Xf for 30 minutes. The precipitate is collected and homogenized into a paste. The paste is cast into a mold, air-dried at 37°C, and the resulting film is washed with water. The washed membrane was then treated with a 0.1 solution of glutaraldehyde in the same manner as in Example 7, and the crosslinked membrane was washed and dried to obtain membrane Xp-2. Example 9 Same as in Example 2. The method of gel formation is modified by degassing and shaping the pre-gel mixture. Before incubating, the mixture is degassed under reduced pressure and placed in the mold. After incubation at 37°C for 16-20 hours, the shaped gelatin was compressed to approximately 1.51 cm to obtain a dense fiber network;
Air dry at 37°C or below. The dried solid is washed, desalted, reshaped, dried, and heated to about 40°C to 1.00°C.
Aging at high temperature of °C increases residual crosslinking and
A product designated as "-2" is obtained.

Example 10 A. Preparation of cross-linked collagen Add 0.02M IJ sodium hydrogen phosphate to the solution at 18-20°C to bring the pH to 7.4, and prepare the fibers for 1-2 hours.
Collagen in solution (3 W/m of bovine atelopeptide collagen in dilute aqueous hydrochloric acid solution)
Reconstitute fibrillar collagen from l solution, pH 1-4)
Ru. 160+/-% of the resulting fibrillar collagen suspension
Add 1.62 ml of 1% glutaraldehyde aqueous solution at pH3. While stirring, gradually add the glutaraldehyde solution to give a final concentration of glutaraldehyde of 0.01%, and the mixture is allowed to react at room temperature for 16 hours before quenching by adding 3M glycine to 0.2M. After quenching for 1 hour, the cross-linked collagen was washed three times with 100 g/ml of buffer containing 0.02 M hydrogen phosphate, 0.13 M NaCJ (pH 7,4), with 1
Centrifuge at 7000 x g for 5-7 minutes. The dynamic viscosity of collagen was measured using a vibrating disk device (Nameter Company (N
ametre Company), Mode/I/7
．． 006 PBD) at a shear rate of about 5000 Equation-1 and was found to be about 700 CP at 22°C. After the final wash and centrifugation, the collagen was resuspended in the buffer and the protein concentration was approximately 3011 Ig/ll.
Preparation of the gel: Mix log/log of the above suspension with 580xl of Tigen Cl and degas the thoroughly mixed ingredients under vacuum.

To the degassed mixture, add 0.2 M Na at room temperature (7).
2 HP O4/ 1.3MNaCJ/ Q, Q9MN
10 sgJ of aOH-containing buffer are added and the mixture is incubated at 37°C overnight. The resulting gel contains 55.5% by weight of crosslinked collagen.

Example 11 The gel of Example 10 is placed in a press and compressed by continuously applying a pressure of about 1.5 atm to obtain a flat collagen fiber network. The resulting network is dried at room temperature, washed with water and air-dried again. The obtained collagen membrane is called cx-2.

Example 12 A crosslinked collagen preparation prepared as in Example 10A was used in a ratio of 1:8 to two parts of collagen in solution (prepared by straining as in Example 1) to form a membrane designated as cx-1. GX for the method of Example 1 to obtain the membrane of Example 3.
A membrane designated as -3 is obtained.

Similarly, Example 4 above except using a 1=8 mixture of cross-linked collagen to CIS collagen as starting material.
．． 5 and 6, px-1, PX-2 and p
Prepare x-3.

A change in the proportion of cross-linked collagen in the product also increases the CI
It is also obtained by varying the ratio of suspension of Example 10A to S.

[Brief explanation of drawings]

FIGS. 1 and 2 are electron micrographs in place of drawings showing the structure of the membrane obtained by the present invention.

Claims (16)

[Claims] (1) If desired, a method for producing a collagen membrane material, which comprises compressing a collagen gel matrix containing up to 90% by weight of crosslinked collagen at a constant pressure to form a fibrous network, and drying the network. . (2) The manufacturing method according to item (1) above, further comprising the step of crosslinking the substance with glutaraldehyde. (3) The method according to item (1) above, in which a gel is formed from collagen solution mixed with a suspension of crosslinked collagen, and the ratio of crosslinked collagen is up to 90% by weight. (4) Optionally, mixing with a suspension of crosslinked collagen;
Collagen in solution containing up to 90% cross-linked collagen by weight is cooled to about 4°C, and the cooled solution is treated with a buffer solution previously cooled to about 4°C to give a pH of about 7 and an ionic strength of about 0.05. The manufacturing method according to item (1) above, wherein a gel is formed by obtaining a mixture having the following properties and incubating the mixture at about 20° C. for about 16 to 20 hours. (5) Optionally mixed with a suspension of cross-linked collagen to bring the proportion of cross-linked collagen up to 90% by weight, collagen in solution is mixed with sufficient salt/buffer solution at room temperature to p.
The method according to item (1) above, wherein a gel is formed by obtaining a mixture having a H of about 7 and a physiological ionic strength and incubating the mixture at about 37° C. for 16 to 20 hours. (6) If desired, the solution collagen which has been mixed with a suspension of cross-linked collagen to make the ratio of cross-linked collagen up to 90% by weight is cooled in advance to about 4°C, and the cooled collagen solution is mixed with the chilled collagen solution at about 4°C in advance. Mix the cooled buffer solution to a pH of about 7.
and an ionic strength of about 0.05, and immediately after mixing, the mixture was heated at about 20° C. for 1 to 2 hours at about 80° C.
The above (1)
) manufacturing method. (7) Crush the collagen gel matrix, centrifuge the crushed matrix at approximately 13,000 x g for approximately 0.5 hours, homogenize the resulting precipitate into a paste, cast the paste, and 1. A method for producing a collagen membrane material, which comprises drying a molded paste at a temperature of 37° C. or lower. (8) The manufacturing method according to item (7) above, further including crosslinking of the membrane with glutaraldehyde. (9) The production method according to item (7) above, wherein a gel is formed from solution collagen mixed with a suspension of cross-linked collagen, and the ratio of cross-linked collagen is up to 90% by weight. (10) If desired, the collagen solution is mixed with a suspension of cross-linked collagen so that the ratio of cross-linked collagen is up to 90% by weight, and is cooled to about 4°C, and the cooled solution is preliminarily heated to about 4°C.
to obtain a mixture having a pH of about 7 and an ionic strength of about 0.05, and the mixture was heated to about 20°C.
The manufacturing method according to item (7) above, wherein a gel is formed by incubating for about 16 to 20 hours. (11) Collagen in solution, optionally mixed with a suspension of cross-linked collagen to bring the proportion of cross-linked collagen up to 90% by weight, is mixed at room temperature with sufficient salt/buffer solution to a pH of about 7 and approximately The method according to item (7) above, wherein a gel is formed by obtaining a mixture having physiological ionic strength and incubating the mixture at about 37°C for 16 to 20 hours. (12) If desired, a collagen solution that has been mixed with a suspension of crosslinked collagen to bring the proportion of crosslinked collagen up to 90% by weight is cooled in advance to about 4°C, and the cooled collagen solution is mixed with a suspension of crosslinked collagen at about 4°C in advance. The buffer solution cooled to
Centrifuge at 000 x g to 13000 x g to obtain supernatant,
Collecting the supernatant and forming a gel by incubating the supernatant at about 20° C. for 16-20 hours.
7) Manufacturing method. (13) A membranous collagen material produced by the production method of item (1) above. (14) A membranous collagen material produced by the production method of item (3) above. (15) A membranous collagen material produced by the production method of item (7) above. (16) A membranous collagen material produced by the production method of item (9) above.